Campylobacter jejuni is a leading bacterial cause of diarrhea worldwide and is increasingly resistant to clinically important antibiotics. The CDC has recently identified drug resistant Campylobacter as a serious antibiotic resistance threat in the United States. Among the known mechanisms involved in antibiotic resistance in Campylobacter, the multidrug efflux system CmeABC (an RND-type efflux transporter) is a significant player and confers resistance to structurally diverse antibiotics and toxic compounds. Additionally, CmeABC plays a critical role in bile resistance and is essential for Campylobacter colonization in the intestinal tract. Most recently it was found that CosR, a response regulator fo oxidative stress, serves as a repressor for cmeABC and modulates the expression of cmeABC in response to oxidative stress. These results strongly suggest that oxidative stress is a previously unidentified physiological signal modulating the function of CmeABC and the CosR-mediated response to oxidative stress may be important for the adaptation of C. jejuni, a microaerobic organism that experiences oxidative stress during transmission and infection of a host. Despite strong preliminary evidence, the detailed mechanisms of the interplay between oxidative stress and the CosR- CmeABC pathway and the role of this interaction in antibiotic and oxidative stress resistance are still unknown. To control antibiotic-resistant Campylobacter, we recently developed a novel strategy that utilizes anti-cmeABC peptide nucleic acid (PNA) to inhibit the expression of cmeABC. This approach was found to be effective in sensitizing Campylobacter to antibiotics in cultures, suggesting that antisense PNA targeting cmeABC is a promising approach for combating antibiotic-resistant Campylobacter. This application is based on these exciting findings and will pursue two Specific Aims i) to determine the mechanisms by which oxidative stress interacts with the CosR-CmeABC pathway and define the role of this interaction in Campylobacter adaptation to antibiotic and oxidative stresses, and ii) to determine the efficiency of anti-cmeABC PNA in preventing the emergence of antibiotic resistant mutants and potentiating antibiotics against Campylobacter in animal models. The proposal is innovative both conceptually and technically as it addresses an emerging theme at the interface between oxidative stress response and antibiotic efflux systems and develops a novel anti-Campylobacter approach by targeting CmeABC. The proposed work will significantly advance the concept that oxidative stress sensing and resistance is a common physiological function of antibiotic efflux systems in bacterial pathogens and will develop an effective mean to extend the utility of existing antibiotics against drug-resistant Campylobacter. Furthermore, the technical platform established in this project can be potentially adapted for the control of other antibiotic resistant pathogens.